The invention relates to a method for the analytical determination of the concentration C of a component of a medical sample, in which a reaction of the sample with reagents leads to a time-dependent change S(t) ("kinetic") in a measured quantity S and the concentration C correlates according to an evaluation curve C=f(X) with an input variable X derived from S(t), in which the calibration curve X=f.sup.-1 (C) inverse to the evaluation curve C=f(X) is not monotonous, so that the same value of the input variable X corresponds on at least two sub-sections of the calibration curve to different values of the concentration C, and the evaluation curve is ambiguous for at east a portion of the possible X values.
In chemical analysis, in particular with the analysis of body fluids such as blood and urine, methods are commonly used which are based on a specific binding reaction of two binding partners exhibiting biological affinity. Immunological interactions in particular are specific binding reactions in this sense, i.e. interactions between antigens or haptens on the one hand and antibodies on the other. Other examples are the lectin-sugar and biotin-streptavidin binding reactions or active substance-receptor interactions. Reference will be made below by way of example, without restricting the universality, to immunological interactions.
Methods of analysis of this kind are distinguished by high specificity and sensitivity of detection. A problem with immunological methods of analysis, which has been known for a long time, consists however in the fact that the evaluation curve C=f(X) is in many cases not unambiguous. This phenomenon is also described as the "(high dose) hook effect". It is observed both with heterogeneous and with homogeneous immunological detection methods.
In the case of heterogeneous one-step sandwich tests, for example, there occurs with a high surplus of the sample antigen a saturation both of the solid-phase-bound antibody and of the labelled antibody. This results in a reduction in the coupling of the labelled antibody with the solid-phase-bound antibody and consequently a reduction in the measurement signal with rising analyte concentration.
An important example of homogeneous methods of analysis based on specific binding reactions are reactions which lead to aggregates of molecules, macromolecules or cross-linkages with small carrier particles, such as latex particles, dextrans, liposomes or metallic suspensions. Said aggregate formation leads to a change in the scattered light behaviour, which may be detected with a suitable physical method of measurement. Determination of the absorbance caused by the turbidity (turbidimetry) and measurement of the light scattering (nephelometry) are in common use.
In such tests the non-monotonous shape of the calibration curve X=f.sup.-1 (C), which leads to the described ambiguity of the input variables X, may be explained by the fact that with high concentrations of the sample antigen the binding sites of the antibodies participating in the reaction are increasingly saturated with antigen. Consequently, with a high surplus of antigen the cross-linking of the primary particles no longer increases, but instead decreases and the turbidity diminishes.
This effect, also described as the "antigen surplus phenomenon", was recognized by Heidelberger and Kendall as long ago as 1935. The non-monotonous calibration curve is therefore often described as the "Heidelberger curve". A more detailed discussion of the problem is contained, for example, in European Patent Specification 0 148 463, to which reference is made here.
This printed publication also contains a detailed discussion of previously known methods for combating the problem of the ambiguity of the Heidelberger curves. Only a short summary of them will be given at this point.
A commonly used method is twin determination with two different sample dilutions. If the test for the diluted sample indicates an apparently higher concentration, the measured sample concentration lies in the falling part of the evaluation curve. Further dilution then usually takes place until a decreasing concentration is obtained for each of two consecutive dilutions.
Instead of the investigation of samples diluted to differing degrees, the concentration of the sample may also be varied by the subsequent addition of sample fluid. A further alternative consists in adding additional antibody after completion of an initial reaction, in order to conclude from the input variable then obtained whether the concentration to be determined lies in the rising or in the falling part of the Heidelberger curve. A disadvantage of each of said methods is that expensive additional handling stages are required. They cannot therefore be used without hesitation on normal automatic analysis units. Moreover, the additional measurement increases the measuring time and hence reduces the sample throughput of the automatic analysis unit.
Attempts are therefore often made to ensure by the use of a large quantity of antibodies that all concentrations of the analyte which occur in practice lie in the "valid measuring range", i.e. in the rising part of the Heidelberger curve. The high concentration of the antibody leads however in the range of relatively small analyte concentrations to a very flat shape of the calibration curve X=f.sup.-1 (C) and consequently to a reduced analytical accuracy. In addition the costs of the test are increased considerably by the large quantity of antibodies. In the case of certain analytes which have to be analysed in very high concentrations or in a very wide concentration range, said method is impossible to implement in practice.
In German Patent Specification 27 24 722 an immunonephelometric method is described, which is based on the determination of the variation in time (kinetics) of the turbidity and is intended to permit without spending any more time an unambiguous assignment of the measured value to the rising or falling part of the calibration curve. In the initial period of the turbidity measurements, end point determinations had been carried out, i.e. use was made as the input variable X of the constant value of the measured quantity S which is obtained asymptotically at the end of the aggregation reaction leading to the turbidity. Some years prior to the filing date of German Patent Specification 27 24 722 it had been proposed that the long measuring time required for the end point determination be reduced by means of a kinetic measurement. Said proposal is based on the finding that the maximum rate of change of the measured quantity S (dS/dt.sub.max), which is also referred to as V.sub.max, may be used as the input variable X, which correlates with the concentration C of the analyte antigen with an accuracy sufficient for the quantitative analysis. Said relation C=f(X) is however also ambiguous. In German Patent Specification 27 24 722 it is proposed that the ambiguity of said evaluation curve be eliminated by means of a function of the measured quantity V.sub.max, which is said to possess characteristically different values according to the respective surplus state. In particular the time up to the occurrence of the maximum rate of change V.sub.max of the turbidity signal or the derivation over time of V.sub.max (dV/dt).sub.max =(d.sup.2 S/dt.sup.2).sub.max is regarded as suitable for discrimination of the sub-sections of the evaluation curve. In this literature source it is noted, however, that the inherently desirable unambiguous determination of an individual concentration value is in practice not possible on this basis, because no individual time value exists at which the antibody surplus curve section lies on one side of said value and the antigen surplus curve section lies on the other side (column 21, lines 61 to 68). This problem is said to be solved by a transformation of coordinates and the introduction of new variables. If an antigen surplus state is found on this basis, the sample is diluted and the measurement repeated.
In order to overcome these disadvantages, it is proposed in European Patent Specification 0 148 463 that the functional relationship between the concentration C, the maximum rate of change of the measurement signal V.sub.max and the time from the start of the reaction up to the occurrence of the maximum reaction velocity t.sub.max be determined empirically with a standard preparation and that both V.sub.max and t.sub.max be measured from the sample, one of the two concentrations determinable from the first of said input variables being selected with the aid of the second input variable.
These known methods assume that reliable assignment to the sub-sections of the ambiguous evaluation curve C=f(X) is possible with the aid of a particular additional input variable derived from S(t). They are consequently applicable only in those cases where such a quantity may be found with sufficient discriminating potential. Moreover, these previously known methods depend on whether the curve of S(t) is determined continuously or at least at very close intervals of time (quasi-continuously). This is not possible in practice with many analytical units, because the latter, by virtue of their construction principle, permit the required turbidimetric or nephelometric measurement only at discrete measuring times with relatively large time intervals between them. This applies in particular to the commonly used analytical units which are equipped with a step-wise rotating reaction rotor, with which measurement of the contents of one of the reactors lined up at the periphery of the rotor is possible only when the latter is located at a measuring station.